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Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay
(2014)
Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.
In engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
The fascinating properties of natural biominerals, such as stiffness combined with toughness, are difficult to mimick in synthetic pathways. Therefore, this work investigated the biosynthesis of biomineralization proteins in different organisms for producing synthetic minerals under the control of a protein phase. The soluble nacre protein perlucin from Haliotis was produced in bacteria but remained insoluble after native extraction. It was partially solubilized when fused to a GFP, which was additionally identified to be a strong inhibitor of calcium carbonate formation. Various forms of synthetic biominerals were obtained in simple calcium carbonate precipitation assays depending on the first ionic interaction partner, assay volume and protein concentrations. In comparison to bacteria as less complex organisms, the biomineralization proteins perlucin, N16N and ovocleidin-17 were also expressed in the plant cell wall of Arabidopsis and Nicotiana, which has not been described in the literature so far. This work demonstrated that N16N-GFP and ovocleidin-17-GFP can be expressed in epidermal leaf cells of Nicotiana. In perlucin-GFP transformed Arabidopsis, the plant morphology changed although neither the protein nor RNA were detected. The expression of foreign biomineralization proteins in biotechnologically relevant plants was demonstrated for the first time, opening routes toward new and improved materials.
Methylsulfone derivatized poly(ethylene) glycol (PEG) macromers can be biofunctionalized with thiolated ligands and cross-linked with thiol-based cross-linkers to obtain bioactive PEG hydrogels for in situ cell encapsulation. Methylsulfonyl-thiol (MS-SH) reactions present several advantages for this purpose when compared to other thiol-based cross-linking systems. They proceed with adequate and tunable kinetics for encapsulation, they reach a high conversion degree with good selectivity, and they generate stable reaction products. Our previous work demonstrated the cytocompatibility of cross-linked PEG-MS/thiol hydrogels in contact with fibroblasts. However, the cytocompatibility of the in situ MS-SH cross-linking reaction itself, which generates methylsulfinic acid as byproduct at the cross-linked site, remains to be evaluated. These studies are necessary to evaluate the potential of these systems for in vivo applications. Here we perform an extensive cytocompatibility study of PEG hydrogels during in situ cross-linking by the methylsulfonyl-thiol reaction. We compare these results with maleimide–thiol cross-linked PEGs which are well established for cell culture and in vivo experiments and do not involve the release of a byproduct. We show that fibroblasts and endothelial cells remain viable after in situ polymerization of methylsulfonyl-thiol gels on the top of the cell layers. Cell viability seems better than after in situ cross-linking hydrogels with maleimide–thiol chemistry. The endothelial cell proinflammatory phenotype is low and similar to the one obtained by the maleimide–thiol reaction. Finally, no activation of monocytes is observed. All in all, these results demonstrate that the methylsulfonyl-thiol chemistry is cytocompatible and does not trigger high pro-inflammatory responses in endothelial cells and monocytes. These results make methylsulfonyl-thiol chemistries eligible for in vivo testing and eventually clinical application in the future.
Unlike genomic alterations, gene expression profiles have not been widely used to refine cancer therapies. We analyzed transcriptional changes in acute myeloid leukemia (AML) cell lines in response to standard first-line AML drugs cytarabine and daunorubicin by means of RNA sequencing. Those changes were highly cell- and treatment-specific. By comparing the changes unique to treatment-sensitive and treatment-resistant AML cells, we enriched for treatment-relevant genes. Those genes were associated with drug response-specific pathways, including calcium ion-dependent exocytosis and chromatin remodeling. Pharmacological mimicking of those changes using EGFR and MEK inhibitors enhanced the response to daunorubicin with minimum standalone cytotoxicity. The synergistic response was observed even in the cell lines beyond those used for the discovery, including a primary AML sample. Additionally, publicly available cytotoxicity data confirmed the synergistic effect of EGFR inhibitors in combination with daunorubicin in all 60 investigated cancer cell lines. In conclusion, we demonstrate the utility of treatment-evoked gene expression changes to formulate rational drug combinations. This approach could improve the standard AML therapy, especially in older patients.
The restoration of neuronal activity after injury or during aging requires neuron repopulation at the site of injury, directional regeneration of new nerves and oriented generation of new synapses. The ECM protein Laminin is abundant in neuronal microenvironment and is known to be involved in directing neuronal migration, differentiation and neurite development. In this thesis, a strategy for in vitro directional neurite growth in soft hydrogels is presented. It is based on the spatiotemporal control of the availability of Laminin adhesive motifs within synthetic hydrogels using light as an external guiding trigger. Different variants of Laminin mimetic peptides containing the IKVAV were selected as ligands to mediate control over axonal growth on biomaterials. The photo- cleavable groups 3-(4,5-dimethoxy-2-nitrophenyl)-2-butanol (DMNPB), 6-nitroveratryl alcohol (NVOC) and 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl] 4-yl)azanediyl)bis(ethan-1-ol) (HANBP) were inserted at the K rest of the peptide to temporally block IKVAV bioactivity. Poly(acrylamide) (PAAm) hydrogel films with varied stiffness from 0.2-70 kPa were used as 2D substrates to study IKVAV-guided directional growth of axons. Two novel acryl monomers carrying methylsulfone (MS) side chains were developed to tune specific coupling of thiol terminated IKVAV to the PAAm gel at physiological conditions. The ability of the photoactivatable IKVAV-containing peptides to trigger and support neurite outgrowth was studied and compared in different cell biology experiments using neural progenitor cells from mouse embryo. The ability of the photoactivatable IKVAV-containing peptides to trigger and support spatial organization of neurons was demonstrated by using masked irradiation. The in-situ light exposure of IK(HANBP)VAV by scanning lasers allowed spatially directed neurite development in 2D cell cultures. In the last part of the Thesis, an attempt to extend the photoactivation strategy to 3D environments was made by using two-photon activatable chromophores. The p-methoxynitrobiphenyl (PMNB) photoremovable group was introduced at aspartic acid residue of RGD sequence, a common adhesive motif used for cell attachment to biomaterials. Degradable hydrogels modified with RGD(PMNB)fC peptide were developed and 3D resolved spatial photoactivation inside the gel using two-photon laser guided migration of fibroblasts L929 within the 3D network was established. These results demonstrate that photoactivatable adhesive peptides can be used for spatiotemporal activation of attachment, migration and directional growth of cells in 2D and 3D cultures and provide a tool to control and pattern cell processes in relevant biomedical applications
o-Nitrobenzyl (oNB)-based polymers have been used as photodegradable materials/surfaces, and responsive biomaterials. While previous studies have mainly focused on the photodegradation of the material by intercalating oNB derivatives in the polymer chains, this thesis pays attention to the utilization of oNB photolysis to tailor the properties of materials, including interface functions, network topology, and bulk properties. To this end, two kinds of oNB based molecules with multifunctional units were designed and synthesized: 2-bromo-N-(2-nitro-3, 4-dihydroxyphenethyl)-2-methylpropanamide (NO2-BDAM, nitrodapomine based initiator) and 2-((2-bromo-2-methylpropanoyl)oxy)ethyl 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy) butanoate (vinyl-oNB-Br, photolabile inimer). In part 1, NO2-BDAM, in combination with Mn2(CO)10 as a visible light-sensitive additive, was used to control the growth and detachment of polymer brushes independently. In part 2, vinyl-oNB-Br was introduced into a double network material for in situ regulating topology from connected double network (c-DN) to disconnected double network (d-DN) by light exposure. In the last part, vinyl-oNB-Br was used to graft and detach polymer brushes from a network, leading to the change of network topology and material toughness. These results contribute to the topic of functionalized oNB based polymer systems and tunable topological polymer networks, providing useful strategies both in chemistry and materials.
Two-photon (2P) activable photocleavable protecting groups (PPGs) can be introduced in polymer networks as photodegradation sites or as blocking groups for active sites, which enable the alternation of mechanical properties and biochemical signals and allow to study consequent cell response in a spatiotemporal controlled manner. So far, the design of high efficient 2P activable hydrogels is challenging. This Thesis presents novel designs of photodegradable hydrogels that contain the 4’-methoxy-4-nitrobiphenyl-3-yleth-2-yl)methyl (PMNB) PPG. PMNB-gels formed under physiological conditions and showed tuneable hydrolytic stability and adequate rate for cell encapsulation. Moreover, PMNB-gels can be photodegraded efficiently upon 2P excitation (λ = 740 nm). Preliminary experiments of PMNB-gels as 4D matrices for the investigation of cell response are presented. In a second part, a 2P-activatable PPGs endowed with an extended π conjugation was demonstrated and introduced to yield the RGD cell adhesive peptide. The targeted peptide is obtained but only in low yield due to its low stability. The results of this Thesis provide new tools for instructing cells in 3D cultures using 2P-activated processes and demonstrate the potential of photochemistry for the realization of 4D biomaterials.
Bacterial growth and metabolic activity are sensitive to the mechanical properties of their environment. Understanding how the 3D spatial confinement regulates the cell behavior is crucial not only for understanding biofilm development but also for the design and safe application of engineered materials containing living cells. This Thesis explores the use of Pluronic-based hydrogels to encapsulate genetically modified Escherichia coli bacteria. Hydrogels with different viscoelastic properties were prepared by mixing Pluronic and Pluronic diacrylate components in different ratios, giving physical hydrogels with variable degree of covalent crosslinking and different mechanical responses. Rheological properties of the hydrogels as well as the growth rate and morphology of the embedded bacterial colonies were characterized. The results provided correlations between material parameters and bacterial cell responses. Further, a bilayer thin film model was developed for long term encapsulation of the organisms, preventing leakage of cells for up to two weeks while maintaining their activity as drug/protein eluting devices or biosensing units. The bacterial bilayer thin films did not elicit significant immune responses in primary immune cells from healthy donors. The results of this Thesis demonstrate the potential of Pluronic-based biohybrid as a suitable and safe prototype for further in vitro and in vivo testing of engineered living material designs.
Regenerative medicine aims to restore damaged cells, tissues, and organs, for which growth factors are vital to stimulate regenerative cellular transformations. Major advances have been made in growth factor engineering and delivery like the development of robust peptidomimetics and controlled release matrices. However, their clinical applicability remains limited due to their poor stability in the body and need for careful regulation of their local concentration to avoid unwanted side-effects. In this study, a strategy to overcome these limitations is explored using engineered living materials (ELMs), which contain live microorganisms that can be programmed with stimuli-responsive functionalities. Specifically, the development of an ELM that releases a pro-angiogenic protein in a light-regulated manner is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a vascular endothelial growth factor peptidomimetic (QK) linked to a collagen-binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. In situ control over the release profiles of the pro-angiogenic protein using light is demonstrated. Finally, it is shown that the released protein is able to bind collagen and promote angiogenic network formation among vascular endothelial cells, indicating the regenerative potential of these ELMs.
